Oligonucleotide-based drugs show promise as a novel form of chemotherapy. Among the hurdles that have to be overcome on the way of applicable nucleic acid therapeutics, inefficient cellular uptake and subsequent release from endosomes to cytoplasm appear to be the most severe ones. Thus, the ability to deliver nucleic acids (e.g., antisense oligonucleotides and their peptide nucleic acid or phosphorodiamidate morpholino analogues, plasmid DNA, RNA such as mRNA, tRNA and siRNA) to specific cell-type offers opportunities to overcome these shortcomings and the potential to develop novel therapeutics for the treatment of many diseases that are difficult to treat effectively with traditional therapies (e.g., hereditary diseases, cancer).
Watson-Crick base-pairing of nucleic acid sequences with key sequences of mRNA targets has been shown to inhibit the ability of these mRNAs to interact with the cellular machinery required for protein synthesis. For example, the interaction of nucleic acid sequences with the initiation codon or splicing sequences of mRNA targets can alter the transcription of these mRNAs and expression of the encoded proteins. In this context, most human genes undergo alternative splicing events, which are triggered by an intricate and highly regulated machinery requiring the sequence-specific binding of several proteins to nuclear pre-mRNAs. Steric interference imparted by RNase H-incompetent nucleic acid analogues complementary to specific pre-mRNA splice sites has been shown to be efficient in re-directing the splicing machinery during assembly of the mature mRNAs. The biomedical relevance of alternative splicing events has been demonstrated through skipping the mutated exons in dystrophin pre-mRNA as a viable approach to the clinical treatment of Duchenne muscular dystrophy.
Steric interference leading to splice correction requires sequence-specific and high affinity binding of nuclease-resistant RNase-H incompetent oligonucleotide analogues complementary to any targeted intron-exon junctions in the precursor mRNA. Negatively charged 2′-O-methyl RNA phosphorothioate sequences and uncharged peptide nucleic acid (PNA) or phosphorodiamidate morpholino (PMO) oligomers were found adequate for inducing splice correcting events. Currently, cationic lipids are the most popular carriers for in vitro cellular transfection of negatively charged DNA/RNA oligonucleotides and their analogues. However, in addition to being cytotoxic and of poor stability in the presence of serum proteins, these carriers cannot be used for the cellular internalization of uncharged PNAs or PMOs. Remarkably, the conjugation of cationic cell penetrating peptides (CPPs) to PNA or PMO oligomers led to pre-mRNA splicing correction activities in mammalian cells. It should however be noted that the covalent attachment of highly cationic CPPs to PNAs or PMOs may be challenging, as it could produce insoluble conjugates or could inhibit the PNA or PMO portion of the conjugates to perform their intended functions. Although this method was successful at eliciting corrective nuclear pre-mRNA splicing events, this approach to the cellular delivery of PNA oligomers faces technical and cost-effectiveness challenges.
It is well documented that RNA interference (RNAi) has broad potential for silencing any gene. To achieve the clinical potential of RNAi, delivery materials are required to transport short negatively charged double-stranded interfering RNAs (siRNAs) to the site of action in the cells of target tissues. A clinically advanced platform for the cellular delivery of siRNAs is based on the use of dynamic polyconjugates (DPC). These conjugates incorporate several components, each intended to play a particular role in the delivery process. Typically, siRNAs are attached to a membrane-disrupting polymer by a hydrolysable disulfide linker; the polymer is shielded by polyethylene glycol (PEG), which is designed to be cleaved in an acidic endosomal environment and expose the membrane-disrupting polymer for endosomal release. In the cytosol, the siRNAs are cleaved from the polymer and trigger the RNAi machinery. DPC systems have been shown effective at silencing two different genes when administered intravenously. However, the complexity of DPC systems creates problems in the scale-up of their manufacturing protocols where tightly controlled mixing steps are required to achieve consistent quality of the polyconjugates.
The delivery of DNA and RNA can be used, under gene therapy settings, to tackle various acquired or heritable diseases, where natural immunity is aberrant or lacking. Gene therapy is based on the underlying principle that disease can be addressed by introduction of exogenous genetic material into somatic cells of patients for the purpose of modulating gene expression of desired proteins.
Shortcomings in the delivery of genetic material arise because of the sensitivity of unprotected genetic material to extracellular enzymes. After cellular entry, the genetic material is subjected to intracellular degradation in endosomal/lysosomal compartments. Unassisted genetic material compartmental release is negligible and the small fraction that may escape is exposed to further cytoplasmic degradation and plagued with poor translocation kinetics. When nuclear entry is required for therapeutic activity, crossing the nuclear membranes represents an additional impediment. Regardless of the desired outcome, the use of natural or artificial gene carrier vector systems are required to overcome the above limitations.
The formation of stable cationic lipids-nucleic acid particles that possess the chemical and biophysical properties required to overcome delivery obstructions is essential for an effective gene delivery process. Complexation of genetic material in the formation of lipoplexes proceeds by electrostatic interactions between cationic vectors and anionic nucleic acids. This process results in the compaction of sub-micrometer-sized particles, each composed of numerous DNA (or RNA) molecules. Effective particle formation sterically protects genetic materials from nucleolytic enzymes, enhances cell permeability/uptake, and increases cytosolic mobility. Complexation of genes is most easily controlled by adjusting lipid:gene weight ratios. Optimal ratios correspond to the amount of vector needed to fully complex the genetic material. Although gene delivery is thought to be improved by use of excessive charge, which enable nucleic acids to achieve transient, yet stable interactions, within the nanoparticle, the use of excessive charge can result in undesired toxicity and binding forces that are too strong for efficient gene unpacking. Thus, desired specific structural properties for each vector type must be considered to allow rational design of nanoparticles with the greatest gene delivery potential.
When all is considered, there is indeed a need in the art for structurally simpler, more efficient and cost effective agents for in vitro cellular transfection of neutral and negatively charged nucleic acids (e.g., plasmid DNA, DNA/RNA oligonucleotides including siRNAs, short hairpin RNAs and microRNAs) and their analogues.